Characterization of the quinolone resistant determining regions in clinical isolates of pneumococci collected in Canada

Abstract

Background: The objective of this study was to examine Streptococcus pneumoniae isolates collected from a longitudinal surveillance program in order to determine their susceptibility to currently used fluoroquinolones and of the frequency and type of mutations in the quinolone-resistant determining regions (QRDRs) of their parC and gyrA genes.

Methods: The Canadian Bacterial Surveillance Network has been collecting clinical isolates of S. pneumoniae from across Canada since 1988. Broth microdilution susceptibility testing was carried out according to the Clinical and Laboratory Standards Institute guidelines. The QRDRs of the parC and gyrA genes were sequenced for all isolates with ciprofloxacin MIC > or = 4 mg/L, and a large representative sample of isolates (N = 4,243) with MIC < or = 2 mg/L.

Results: A total of 4,798 out of 30,111 isolates collected from 1988, and 1993 to 2007 were studied. Of those isolates that were successfully sequenced, 184 out of 1,032 with mutations in parC only, 11 out of 30 with mutations in gyrA only, and 292 out of 298 with mutations in parC and gyrA were considered resistant to ciprofloxacin (MIC > or = 4 mg/L). The most common substitutions in the parC were at positions 137 (n = 722), 79 (n = 209), and 83 (n = 56), of which substitutions at positions 79 and 83 were associated with 4-fold increase in MIC to ciprofloxacin, whereas substitutions at position 137 had minimal effect on the ciprofloxacin MIC. A total of 400 out of 622 isolates with Lys-137 parC mutation belonged to serotypes 1, 12, 31, 7A, 9V, 9N and 9L, whereas only 49 out of 3064 isolates with no mutations belonged to these serotypes. Twenty-one out of 30 isolates with substitutions at position 81 of the gyrA gene had an increased MIC to ciprofloxacin. Finally, we found that isolates with mutations in both parC and gyrA were significantly associated with increased MIC to fluoroquinolones.

Conclusions: Not all mutations, most frequently Lys-137, found in the QRDRs of the parC gene of S. pneumoniae is associated with an increased MIC to fluoroquinolones. The high prevalence of Lys-137 appears to be due to its frequent occurrence in common serotypes.

Figure 1

Distribution of ciprofloxacin MICs in S. pneumoniae isolates with a parC only amino acid substitutions. The figure shows that pneumococcal isolates with substitution at either position 79 or 83 of parC have four-fold increased ciprofloxacin MIC compared to isolates with no mutations in parC. On the other hand, substitution at position 137 of parC does not appear to increase ciprofloxacin MIC.

Figure 2

Distribution of pneumococcal isolates with amino acid substitutions in parC and gyrA against ciprofloxacin MIC. The figure shows that isolates with amino acid substitutions at positions 79 of parC and 81 of gyrA or at positions 79 of parC and 85 of gyrA have 32-fold higher ciprofloxacin MIC compared to isolates with no mutation in parC and gyrA. Similarly, substitutions at positions 83 of parC and 81 of gyrA result in 16-fold increase in ciprofloxacin MIC.